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Cellular uptake of intracerebrally administered oligodeoxynucleotides in mouse brain
ELSEVIER
Regulatory Peptides 59 (1995) 143-149
Cellular uptake of intracerebrally administered oligodeoxynucleotides in mouse brain Sonoko Ogawa *, Harold E. Brown, Hirotaka J. Okano, Donald W. Pfaff Laboratory of Neurobiology and Behavior, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA Received 26 April 1995; accepted 27 June 1995
Abstract
Intracerebral diffusion, cellular uptake and intracellular localization of oligodeoxynucleotides (ODN) after their microinjection in mouse brain:~ were examined. Using either tetramethylrhodamine-5-(and -6)-isothiocyanate (TRITC)- or .,/_33p ATP-labeled ODNs, it was found that both phosphodiester ODNs (D-ODN) and phosphorothioate ODNs (S-ODN) quickly diffused (up to about 500 /xm) and were taken up by many cells around the injection site as early as 15 min after administration. Fluorescence labeling intensity and silver grain accumulation of D-ODNs were greatly reduced by 4 h after injection, whereas those of S-ODNs were stable beyond at least 8 h after injection. Most of labeled ODNs were found in neuronal cells as identified by immunocytochemistry for neurofilament, NF 200, and to a much lesser extent in astrocytic cells as identified by immunocytochemistry for glial fibrillary acidic protein. Keywords: Antisense oligodeoxynucleotide; Hypothalamus; Phosphorothioate oligonucleotide; Phosphodiester oligonucleotide; GFAP
1. Introduction
The antisense D N A / R N A method has been widely used in behavioral studies in the last two to three years as a new and powerful tool to demonstrate the role of certain molecular processes in the regulation of a specific behavior (see reviews, [1,2]). A number of studies have shown that intracerebroventricular (i.c.v.), site-specific intracerebral or intrathecal administrations of antisense D N A / R N A can modify the occurrence of various kinds of behaviors by blocking the synthesis of the targeted gene products of in vivo neuronal systems (e.g., [3-9]). Relatively little has been described, however, regard-
* Corresponding author. Fax: + 1 (212) 3278664.
ing the intracerebral spread, time course of cellular uptake, intracellular distribution and stability of antisense oligodeoxynucleotides (ODN) administered intracerebrally or intracerebroventricularly. Whitesell et al. [10] compared the distribution of labeled cells after bolus or continuous i.c.v, administration of fluorescein-labeled phosphorothioate ODN (S-ODN), which was proved to be much more stable than unmodified phosphodiester (D-ODN) in cerebrospinal fluid in vivo rat brains. They reported that after bolus injections, labeled cells could be detected mainly in the ependymal surfaces and markedly decreased deeper than the ependyma whereas labeled cells were more widely distributed after continuous i.c.v, infusion of ODN with an osmotic minipump for a week. On the other hand, after intracerebral administration both D-ODN [11] and S-ODN [12]
0167-0115/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0167-01 1 5 ( 9 5 ) 0 0 0 9 6 - 8
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s. Ogawaet al./ RegulatoryPeptides59 (1995) 143-149
can be quickly taken up by many cells around the injection site in rat brains. Details of time course of cellular uptake and intracerebral distribution, however, remain to be determined. For many behavioral studies, site-specific intracerebral administration of antisense ODN would be preferred over i.c.v, injection, in order to block the expression of specific genes in discrete brain regions. In the present study, therefore, we aimed to examine diffusion, time course of cellular uptake and intracellular localization of intracerebrally administered antisense ODNs.
2. Materials and methods
2.1. Animals Female Swiss Webster albino mice, (SW)fBR, were purchased from Taconic Labs (Germantown, NY, USA) at the age of 7-8 weeks either as intact or ovariectomized. They were maintained on a 12/12 h light/dark cycle (lights on 10.30 h), at a constant temperature (25°C). Food and water were available ad libitum.
2.2. Preparation of oligonucleotides Two types of ODNs, unmodified phosphodiester ODNs (D-ODN; 15mer) and modified phosphorothioate ODN (S-ODN; 15mer), were used. They were antisense sequences against the translation start site of the B-form of murine progesterone receptor mRNA ( - 6 to + 9; relative to the translation initiation site; [13]). The sequence was 5'-CTCAGTCATGACGAC-3'. For experiments with rhodamine-labeled ODNs (see below), C6 amino-linked (at 5'-end) ODNs were used. All ODNs were synthesized by Oligos Etc Inc (Wilsonville, OR, USA).
2.3. Labeling of oligonucleotides C6 amino-linked ODNs were labeled with tetramethylrhodamine-5-(and -6)-isothiocyanate (TRITC; Research Organics, INC, Cleveland, OH, USA) by incubating 100 /xg of ODN (21 nmol) with 86 nmol TRITC in 10/zl of 0.1 M sodium carbonate, pH 9.0, for 16 h at 25°C. Labeled ODNs were then separated from free TRITC by gel filtration, using Sephadex
G-25. They were lyophilized to dryness and reconstituted in vehicle to get desired concentrations of DNA for infusion. The purity of the labeled ODNs and the absence of free TRITC were checked by PAGE (20% polyacrylamide containing 7 N urea). For some experiments, ODNs were also 5'-endlabeled with ,y_33p A T P (NEN Research Products, Boston, MA, USA) using T4 polynucleotide kinase (Promega, Madison, WI, USA) in buffer containing 100 mM Tris, pH 7.5, 20 mM MgCI 2, 10 mM DTI', 0.2 mM spermidine and 0.2 mM EDTA at 37°C for 30 min. Labeled ODNs were gel filtrated with Sephadex G-25, lyophilized dry, and reconstituted in vehicle with different concentrations. The purity of the labeled ODNs was confirmed by PAGE (20% polyacrylamide containing 7 N urea).
2.4. Infusion of oligonucleotides Animals were anesthetized with chloropent (1 m g / 1 0 g, i.p.) and stereotaxically injected with ODNs through a 32 G needle. Oligonucleotides were injected dorsal to the ventromedial nucleus: AP = Bregma - 1 . 4 to - 1 . 5 mm, ML = 0.5 mm, DV = - 5 . 0 to - 5 . 1 mm. 500 ng of TRITC-labeled ODNs or 1 • 10 6 cpm of 33p-labeled ODNs dissolved in 100 nl or 200 nl of saline were infused over 5 min and the injection needle remained on site for 5 min. Animals were then sacrificed after several different survival times (between 5 min and 24 h).
2.5. Preparation and examination of brain tissue sections Animals infused with TRITC-labeled ODNs were deeply anesthetized and perfused transcardially with 100 mM phosphate-buffered saline (PBS) containing 0.1% heparin, pH 7.2 and 4% paraformaldehyde in 100 mM phosphate buffer (PB), pH 7.2. Brains were removed and stored overnight at 4°C in PBS containing 30% sucrose. Brains were cut at 25 /zm on a cryostat and sections were thaw mounted onto gelatin-coat slides. Slides were then dehydrated and coverslipped for examination with a fluorescent microscope. To examine intracellular localization of TRITC labelling, some adjacent sections were treated with Hoechst 33342 for nuclear chromatin straining without dehydration steps.
s. Ogawa et al./ Regulatory Peptides 59 (1995) 143-149
Animals infused with 33p-labeled ODNs were sacrificed and brains were immediately removed, frozen and cut at 10 txm on a cryostat. Sections were thaw mounted on slides, fixed with 4% paraformaldehyde in 10 mM phosphate-buffered saline (PBS; pH 7.2), washed with a gradient of SSC, dehydrated and either exposed to Kodak scientific imaging fdms (X-OMAT, AR) or dipped in emulsion (NTB3) for autoradiography. 2.6. Immunocytochemistry for NF200 and GFAP
Immunocytochemistry was performed in brain tissues infused with labeled S-ODNs for staining of either neuronal cells or glial cells using polyclonal antibodies (Sigma Immunochemicals, St. Louis, MO, USA) for neurofilament 200 (NF200), or glial fibrillary acidic protein (GFAP), respectively. Sections obtained from brains infused with TRITC-labeled ODNs were incubated with the primary antibodies for 1 h at 37°C, washed in PBS, and then incubated with fluorescein-conjugated secondary antibody (Vector, Burlingame, CA, USA) for 1 h at 37°C. Sections obtained fiom brains infused with 33p. labeled ODNs were fixed, quenched with hydrogen peroxide and blocked with gelatin and normal goat serum. They were incubated with (1) the primary antibodies for 2 days at 4°C or 2 h at 25°C, (2) the biotinylated goat anti-rabbit antibody (Vector) for 2 h at 25°C and (3) the avidin-biotin-complex (Vectastain ABC Elite kit, Vector) for 1 h at 25°C. Sections were rinsed (3 × 5 min) between steps with 10 mM PBS, pH 7.2, which also was used for dilution of all the reagents. They were then treated with diaminobenzidine and hydrogen peroxide (0.03%). After rinsing with PBS, they were dipped in emulsion for autoradiography.
3. Results
It was found that intracerebrally administered ODNs quickly diffused and were taken up by many cells around the injection site. Rostro-caudal and medial-lateral diffusion was about 500 /~m while a relatively large dorsal diffusion along the infusion needle track was often observed. As early as 15 min after infusion, cellular uptake of both TRITC- (Fig.
145
1A and D) and 33p_ (Fig. 2A and C) labeled ODNs was observed. TRITC-labeling was found in cytoplasm, nucleus and to a lesser extent in cellular processes. Occasionally, cytoplasmic staining exhibited a punctate pattern. On the other hand, the nucleolus was often found unlabeled. Although there were no apparent differences in the number of labeled cells between D-ODN and S-ODN infusions at 15 min (Fig. 1A and D), labeling intensity was always higher with S-ODN. With D-ODN infusion, labeling intensity and number of labeled cells greatly decreased at 4 h (Fig. 1B) and very few labeled cells were found at 16 h (Fig. 1C). In contrast, intensely labeled cells were found at 30 min (Fig. 1E), 4 h (Fig. 1F) and 8 h (not shown) in the brains infused with S-ODN. Labeling intensity and number of labeled cells then decreased by 16 h (Fig. 1G) although there were still brightly labeled cells. There were only a few, more lightly labeled cells at 24 h (Fig. 1H) and almost no labeled ODNs were detected at 48 h (Fig. 1I). Similar changes in cellular uptake of ODNs after infusion were observed with 33p_ labeled ODNs. At 4 h after infusion, fewer labeled cells were observed in D-ODN (Fig. 2B) infused brains compared to S-ODN infused brains (Fig. 2D). It was also found that cellular accumulation of grains in 33P-labeled ODN-infused sections was greatly reduced by mixing labeled ODNs with unlabeled ODNs. Most of 33p-labeled ODNs were found in NF200 immunopositive cells (Fig. 3A). Both 33p. (Fig. 3B) and TRITC-(not shown) labeled ODNs were also found in a small number of GFAP immunoreactive cells, but to a much less extent than in neuronal cells.
4. Discussion
It was found in the present study that labeled ODNs rapidly diffused and were taken up by many cells after a single intrahypothalamic administration. These findings are contrasted with those found after a bolus i.c.v, infusion of fluorescein-labeled S-ODNs in rat brains [10], which showed rather poor tissue penetration at 30 min. With a bolus injection of TRITC-labaled S-ODNs into the lateral ventricles of mouse brains, we also found that very few labeled neurons were detected (unpublished data). In the
"~
]
L
/ °
-:
Fig. 1. Photomicrographs showing the time course of cellular uptake of TRITC-labeled D- and S-ODNS (15mer) after infusions into mouse brains. Bar is 50 pm.
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g~
S. Ogawa et al. /Regulatory Peptides 59 (1995) 143-149
present study the numbers of cells taking up TRITC-labeled D-ODNs and S-ODNs were not different immediately after injection (15 min), although labeled D-ODNs were markedly reduced by 4 h, as expected from their relative instability reported in vitro [14,15]. These findings suggest that, even with a single injection, i-)-ODNs can reach most neurons in the vicinity of the injection site. This is again contrasted with a bolus i.c.v, injection of D-ODNs, in which it is unlikely that D-ODNs can reach most target cells: it has been shown that D-ODNs are quickly degraded in~ CSF in vivo [10] even though they are rather stable in CSF in vitro [2]. In spite of extensive cellular uptake of both D-ODNs and SODNs after site-specific intracerebral infusion, however, it might be necessary to use repeated injections to more effectively block the synthesis of the target gene products, especially for constitutively active ones, with antisense ODNs. In contrast, inhibition of
147
rapidly inducible gene products can be relatively easily achieved by a single injection of antisense ODNs: e.g., inhibition of estrogen-inducible progesterone receptor synthesis by antisense ODNs [1,9,16]. Both cytoplasmic and nuclear staining were observed after infusion of TRITC-labeled ODNs: in a study with a confocal microscope, it was also confirmed that TRITC-labeled ODNs were indeed inside the cell (unpublished data). There was an unlabeled area in some of the labeled cells (e.g., Fig. 1D), and this was determined to be the nucleolus by staining TRITC-labeled cells with Hoechst 33342. Nuclear uptake of fluorescein-labeled S-ODNs was also reported in the neostriatum at 4 h after a bolus intracerebral infusions to rat brain [12]. Nuclear uptake of ODNs and efficiencies of antisense ODNs in vitro are known to be enhanced by encapsulating ODNs with cationic lipids [17]. It remains to be determined whether this is the case in vivo application of anti-
Fig. 2. Photomicrograph,;showing the time course of cellular uptake of ~3P-labeled D- and S-ODNs (15mer) after infusions to mouse brains. (A) D-ODN, 15 rain, (B) D-ODNs at 4 h, (C) S-ODNs at 15 min, (D) S-ODNs at 4 h. Bar is 100 p~m.
148
S. Ogawa et al. /Regulatory Peptides 59 (1995) 143-149
Fig. 3. Photomicrographs showing uptake of ~3p-labeled S-ODNs in NF200 (A) or GFAP (B) immunoreactive cells at 4 h after intracerebral infusions. 33P-labeled ODNs were found more frequently in NF200 immuno-positive cells (A), and to a much smaller extent in GFAP immunoreactive cells (B). Bar is 25 /zm. Arrows indicate double-labeled cells.
S. Ogawa et al. /Regulatory Peptides 59 (1995) 143-149
sense ODNs, although it appeared that uptake by the nucleus and nucleolu.,; was more evident with cationic lipid encapsulated ODNs in our preliminary studies (unpublished data). Finally, it was found that ODNs were taken up most avidly by neuronal cells while, to a much smaller extent, both TRITC labeling and silver grain accumulation could he observed in astrocytic cells. These results provide supportive evidence for physiologically significant antisense ODN action on nerve cells.
[8]
[9]
[10]
References [1] Ogawa, S. and Pfaff. D.W., Application of antisense DNA method for the stud) of molecular bases of brain function and behavior, Behav. Genet. (1995) (in press). [2] Wahlestedt, C., Antisense oligonucleotide strategies in neuropharmacology, Trends Pharmacol. Sci., 15 (1994) 42-46. [3] McCarthy, M.M., Kle.opoulos, S.P., Mobbs, C.V. and Pfaff, D.W., Infusion of antisense oligodeoxynucleotides to the oxytocin receptor in the ventromedial hypothalamus reduces estrogen-induced sexual receptivity and oxytocin receptor binding in the female rat, Neuroendocrinology, 59 (1994) 432-440. [4] Standifer, K.M., Chien, C.C., Wahlestedt, C., Brown, G.P. and Pasternak, G.W., Selective loss of delta opioid analgesia and binding by antisense oligodeoxynucleotides to a delta opioid receptor, Neuron, 12 (1994) 805-810. [5] McCarthy, M.M., Masters, D.B., Rimvall, K., SchwartzGiblin, S. and Pfaff, D.W., Intracerebral administration of antisense oligodeoxynucleotides to GAD(65) and GAD(67) mRNAs modulate reproductive behavior in the female rat, Brain Res., 636 (1994) 209-220. [6] Akabayashi, A., Wahlestedt, C., Alexander, J.T. and Leibowitz, S.F., Specific inhibition of endogenous neuropeptide Y synthesis in arcuate nucleus by antisense oligonucleotides suppresses feeding be]aavior and insulin secretion, Mol. Brain Res., 21 (1994) 55-61. [7] McCarthy, M.M., Schleuker, E.H. and Pfaff, D.W., Enduring consequences of neonatal treatment with antisense
[11]
[12]
[13]
[14]
[15]
[16]
[17]
149
oligodeoxynucleotides to estrogen receptor messenger ribonucleic acid on sexual differentiation of rat brain, Endocrinology, 133 (1993) 433-439. Mani, S.K., Allen, J.M.C., Clark, J.H., Blaustein, J.D. and O'Malley, B.W., Convergent pathways for steroid hormoneand neurotransmitter-induced rat sexual behavior, Science, 265 (1994) 1246-1249. Ogawa, S., Olazabal, U.E., Parhar, I.S. and Pfaff, D.W., Effects of intrahypothalamic administration of antisense DNA for progesterone receptor mRNA on reproductive behavior and progesterone receptor immunoreactivity in female rat, J. Neurosci., 14 (1994) 1766-1774. Whitesell, L., Geselowitz, D., Cbavany, C., Fahmy, B., Walbridge, S., Alger, J.R. and Neckers, L.M., Stability, clearance, and disposition of intraventricularly administered oligodeoxynucleotides -implications for therapeutic application within the central nervous system, Proc. Natl. Acad. Sci. USA, 90 (1993) 4665-4669. McCarthy, M,M., Brook, P.J., Pfaus, J.G., Brown, H.E., Flanagan, L.M., Schwarz-Giblin, S. and Pfaff, D.W., Antisense oligodeoxynucleotides in behavioral neuroscience, Neuroprotocols, 2 (1993) 67-74. Sommer, W., Bjelke, B., Ganten, D. and Fuxe, K., Antisense oligonucleotide to c-fos induces ipsilateral rotational behaviour to d-amphetamine, Neuroreport, 5 (1993) 227-280. Hagihara, K., Wu-Peng, X.S., Funabashi, T., Kato, J. and Pfaff, D.W., Nucleic acid sequence and DNAse hypersensitive sites of the 5'region of the mouse progesterone receptor gene, Biochem. Biophys. Res. Commun., 205 (1994) 10931101. Akhtar, S., Kole, R. and Juliano, R.L., Stability of antisense DNA oligodeoxynucleotide analogs in cellular extracts and sera, Life Sci., 49 (1991) 1793-1801. Akhtar, S., Shoji, Y. and Juliano, R.L., Pharmaceutical aspects of the biological stability and membrane transport characteristics of antisense oligonucleotides, Gene Regulation, 1 (1992) 133-143. Pollio, G., Xue, P., Zanisi, M., Nicolin, A. and Maggi, A., Antisense oligonucleotide blocks progesterone-induced lordosis behavior in ovariectomized rats, Mol. Brain Res., 19 (1993) 135-139. Bennett, C.F., Chiang, M.Y., Chart, H.D., Shoemaker, J. and MirabeUi, C.K., Cationic lipids enhance cellular uptake and activity of phosphorothioate antisense oligonucleotides, Mol. Pharmacol., 41 (1992) 1023-1033.
Regulatory Peptides 59 (1995) 143-149
Cellular uptake of intracerebrally administered oligodeoxynucleotides in mouse brain Sonoko Ogawa *, Harold E. Brown, Hirotaka J. Okano, Donald W. Pfaff Laboratory of Neurobiology and Behavior, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA Received 26 April 1995; accepted 27 June 1995
Abstract
Intracerebral diffusion, cellular uptake and intracellular localization of oligodeoxynucleotides (ODN) after their microinjection in mouse brain:~ were examined. Using either tetramethylrhodamine-5-(and -6)-isothiocyanate (TRITC)- or .,/_33p ATP-labeled ODNs, it was found that both phosphodiester ODNs (D-ODN) and phosphorothioate ODNs (S-ODN) quickly diffused (up to about 500 /xm) and were taken up by many cells around the injection site as early as 15 min after administration. Fluorescence labeling intensity and silver grain accumulation of D-ODNs were greatly reduced by 4 h after injection, whereas those of S-ODNs were stable beyond at least 8 h after injection. Most of labeled ODNs were found in neuronal cells as identified by immunocytochemistry for neurofilament, NF 200, and to a much lesser extent in astrocytic cells as identified by immunocytochemistry for glial fibrillary acidic protein. Keywords: Antisense oligodeoxynucleotide; Hypothalamus; Phosphorothioate oligonucleotide; Phosphodiester oligonucleotide; GFAP
1. Introduction
The antisense D N A / R N A method has been widely used in behavioral studies in the last two to three years as a new and powerful tool to demonstrate the role of certain molecular processes in the regulation of a specific behavior (see reviews, [1,2]). A number of studies have shown that intracerebroventricular (i.c.v.), site-specific intracerebral or intrathecal administrations of antisense D N A / R N A can modify the occurrence of various kinds of behaviors by blocking the synthesis of the targeted gene products of in vivo neuronal systems (e.g., [3-9]). Relatively little has been described, however, regard-
* Corresponding author. Fax: + 1 (212) 3278664.
ing the intracerebral spread, time course of cellular uptake, intracellular distribution and stability of antisense oligodeoxynucleotides (ODN) administered intracerebrally or intracerebroventricularly. Whitesell et al. [10] compared the distribution of labeled cells after bolus or continuous i.c.v, administration of fluorescein-labeled phosphorothioate ODN (S-ODN), which was proved to be much more stable than unmodified phosphodiester (D-ODN) in cerebrospinal fluid in vivo rat brains. They reported that after bolus injections, labeled cells could be detected mainly in the ependymal surfaces and markedly decreased deeper than the ependyma whereas labeled cells were more widely distributed after continuous i.c.v, infusion of ODN with an osmotic minipump for a week. On the other hand, after intracerebral administration both D-ODN [11] and S-ODN [12]
0167-0115/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0167-01 1 5 ( 9 5 ) 0 0 0 9 6 - 8
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s. Ogawaet al./ RegulatoryPeptides59 (1995) 143-149
can be quickly taken up by many cells around the injection site in rat brains. Details of time course of cellular uptake and intracerebral distribution, however, remain to be determined. For many behavioral studies, site-specific intracerebral administration of antisense ODN would be preferred over i.c.v, injection, in order to block the expression of specific genes in discrete brain regions. In the present study, therefore, we aimed to examine diffusion, time course of cellular uptake and intracellular localization of intracerebrally administered antisense ODNs.
2. Materials and methods
2.1. Animals Female Swiss Webster albino mice, (SW)fBR, were purchased from Taconic Labs (Germantown, NY, USA) at the age of 7-8 weeks either as intact or ovariectomized. They were maintained on a 12/12 h light/dark cycle (lights on 10.30 h), at a constant temperature (25°C). Food and water were available ad libitum.
2.2. Preparation of oligonucleotides Two types of ODNs, unmodified phosphodiester ODNs (D-ODN; 15mer) and modified phosphorothioate ODN (S-ODN; 15mer), were used. They were antisense sequences against the translation start site of the B-form of murine progesterone receptor mRNA ( - 6 to + 9; relative to the translation initiation site; [13]). The sequence was 5'-CTCAGTCATGACGAC-3'. For experiments with rhodamine-labeled ODNs (see below), C6 amino-linked (at 5'-end) ODNs were used. All ODNs were synthesized by Oligos Etc Inc (Wilsonville, OR, USA).
2.3. Labeling of oligonucleotides C6 amino-linked ODNs were labeled with tetramethylrhodamine-5-(and -6)-isothiocyanate (TRITC; Research Organics, INC, Cleveland, OH, USA) by incubating 100 /xg of ODN (21 nmol) with 86 nmol TRITC in 10/zl of 0.1 M sodium carbonate, pH 9.0, for 16 h at 25°C. Labeled ODNs were then separated from free TRITC by gel filtration, using Sephadex
G-25. They were lyophilized to dryness and reconstituted in vehicle to get desired concentrations of DNA for infusion. The purity of the labeled ODNs and the absence of free TRITC were checked by PAGE (20% polyacrylamide containing 7 N urea). For some experiments, ODNs were also 5'-endlabeled with ,y_33p A T P (NEN Research Products, Boston, MA, USA) using T4 polynucleotide kinase (Promega, Madison, WI, USA) in buffer containing 100 mM Tris, pH 7.5, 20 mM MgCI 2, 10 mM DTI', 0.2 mM spermidine and 0.2 mM EDTA at 37°C for 30 min. Labeled ODNs were gel filtrated with Sephadex G-25, lyophilized dry, and reconstituted in vehicle with different concentrations. The purity of the labeled ODNs was confirmed by PAGE (20% polyacrylamide containing 7 N urea).
2.4. Infusion of oligonucleotides Animals were anesthetized with chloropent (1 m g / 1 0 g, i.p.) and stereotaxically injected with ODNs through a 32 G needle. Oligonucleotides were injected dorsal to the ventromedial nucleus: AP = Bregma - 1 . 4 to - 1 . 5 mm, ML = 0.5 mm, DV = - 5 . 0 to - 5 . 1 mm. 500 ng of TRITC-labeled ODNs or 1 • 10 6 cpm of 33p-labeled ODNs dissolved in 100 nl or 200 nl of saline were infused over 5 min and the injection needle remained on site for 5 min. Animals were then sacrificed after several different survival times (between 5 min and 24 h).
2.5. Preparation and examination of brain tissue sections Animals infused with TRITC-labeled ODNs were deeply anesthetized and perfused transcardially with 100 mM phosphate-buffered saline (PBS) containing 0.1% heparin, pH 7.2 and 4% paraformaldehyde in 100 mM phosphate buffer (PB), pH 7.2. Brains were removed and stored overnight at 4°C in PBS containing 30% sucrose. Brains were cut at 25 /zm on a cryostat and sections were thaw mounted onto gelatin-coat slides. Slides were then dehydrated and coverslipped for examination with a fluorescent microscope. To examine intracellular localization of TRITC labelling, some adjacent sections were treated with Hoechst 33342 for nuclear chromatin straining without dehydration steps.
s. Ogawa et al./ Regulatory Peptides 59 (1995) 143-149
Animals infused with 33p-labeled ODNs were sacrificed and brains were immediately removed, frozen and cut at 10 txm on a cryostat. Sections were thaw mounted on slides, fixed with 4% paraformaldehyde in 10 mM phosphate-buffered saline (PBS; pH 7.2), washed with a gradient of SSC, dehydrated and either exposed to Kodak scientific imaging fdms (X-OMAT, AR) or dipped in emulsion (NTB3) for autoradiography. 2.6. Immunocytochemistry for NF200 and GFAP
Immunocytochemistry was performed in brain tissues infused with labeled S-ODNs for staining of either neuronal cells or glial cells using polyclonal antibodies (Sigma Immunochemicals, St. Louis, MO, USA) for neurofilament 200 (NF200), or glial fibrillary acidic protein (GFAP), respectively. Sections obtained from brains infused with TRITC-labeled ODNs were incubated with the primary antibodies for 1 h at 37°C, washed in PBS, and then incubated with fluorescein-conjugated secondary antibody (Vector, Burlingame, CA, USA) for 1 h at 37°C. Sections obtained fiom brains infused with 33p. labeled ODNs were fixed, quenched with hydrogen peroxide and blocked with gelatin and normal goat serum. They were incubated with (1) the primary antibodies for 2 days at 4°C or 2 h at 25°C, (2) the biotinylated goat anti-rabbit antibody (Vector) for 2 h at 25°C and (3) the avidin-biotin-complex (Vectastain ABC Elite kit, Vector) for 1 h at 25°C. Sections were rinsed (3 × 5 min) between steps with 10 mM PBS, pH 7.2, which also was used for dilution of all the reagents. They were then treated with diaminobenzidine and hydrogen peroxide (0.03%). After rinsing with PBS, they were dipped in emulsion for autoradiography.
3. Results
It was found that intracerebrally administered ODNs quickly diffused and were taken up by many cells around the injection site. Rostro-caudal and medial-lateral diffusion was about 500 /~m while a relatively large dorsal diffusion along the infusion needle track was often observed. As early as 15 min after infusion, cellular uptake of both TRITC- (Fig.
145
1A and D) and 33p_ (Fig. 2A and C) labeled ODNs was observed. TRITC-labeling was found in cytoplasm, nucleus and to a lesser extent in cellular processes. Occasionally, cytoplasmic staining exhibited a punctate pattern. On the other hand, the nucleolus was often found unlabeled. Although there were no apparent differences in the number of labeled cells between D-ODN and S-ODN infusions at 15 min (Fig. 1A and D), labeling intensity was always higher with S-ODN. With D-ODN infusion, labeling intensity and number of labeled cells greatly decreased at 4 h (Fig. 1B) and very few labeled cells were found at 16 h (Fig. 1C). In contrast, intensely labeled cells were found at 30 min (Fig. 1E), 4 h (Fig. 1F) and 8 h (not shown) in the brains infused with S-ODN. Labeling intensity and number of labeled cells then decreased by 16 h (Fig. 1G) although there were still brightly labeled cells. There were only a few, more lightly labeled cells at 24 h (Fig. 1H) and almost no labeled ODNs were detected at 48 h (Fig. 1I). Similar changes in cellular uptake of ODNs after infusion were observed with 33p_ labeled ODNs. At 4 h after infusion, fewer labeled cells were observed in D-ODN (Fig. 2B) infused brains compared to S-ODN infused brains (Fig. 2D). It was also found that cellular accumulation of grains in 33P-labeled ODN-infused sections was greatly reduced by mixing labeled ODNs with unlabeled ODNs. Most of 33p-labeled ODNs were found in NF200 immunopositive cells (Fig. 3A). Both 33p. (Fig. 3B) and TRITC-(not shown) labeled ODNs were also found in a small number of GFAP immunoreactive cells, but to a much less extent than in neuronal cells.
4. Discussion
It was found in the present study that labeled ODNs rapidly diffused and were taken up by many cells after a single intrahypothalamic administration. These findings are contrasted with those found after a bolus i.c.v, infusion of fluorescein-labeled S-ODNs in rat brains [10], which showed rather poor tissue penetration at 30 min. With a bolus injection of TRITC-labaled S-ODNs into the lateral ventricles of mouse brains, we also found that very few labeled neurons were detected (unpublished data). In the
"~
]
L
/ °
-:
Fig. 1. Photomicrographs showing the time course of cellular uptake of TRITC-labeled D- and S-ODNS (15mer) after infusions into mouse brains. Bar is 50 pm.
I;(
4~
L~
g~
S. Ogawa et al. /Regulatory Peptides 59 (1995) 143-149
present study the numbers of cells taking up TRITC-labeled D-ODNs and S-ODNs were not different immediately after injection (15 min), although labeled D-ODNs were markedly reduced by 4 h, as expected from their relative instability reported in vitro [14,15]. These findings suggest that, even with a single injection, i-)-ODNs can reach most neurons in the vicinity of the injection site. This is again contrasted with a bolus i.c.v, injection of D-ODNs, in which it is unlikely that D-ODNs can reach most target cells: it has been shown that D-ODNs are quickly degraded in~ CSF in vivo [10] even though they are rather stable in CSF in vitro [2]. In spite of extensive cellular uptake of both D-ODNs and SODNs after site-specific intracerebral infusion, however, it might be necessary to use repeated injections to more effectively block the synthesis of the target gene products, especially for constitutively active ones, with antisense ODNs. In contrast, inhibition of
147
rapidly inducible gene products can be relatively easily achieved by a single injection of antisense ODNs: e.g., inhibition of estrogen-inducible progesterone receptor synthesis by antisense ODNs [1,9,16]. Both cytoplasmic and nuclear staining were observed after infusion of TRITC-labeled ODNs: in a study with a confocal microscope, it was also confirmed that TRITC-labeled ODNs were indeed inside the cell (unpublished data). There was an unlabeled area in some of the labeled cells (e.g., Fig. 1D), and this was determined to be the nucleolus by staining TRITC-labeled cells with Hoechst 33342. Nuclear uptake of fluorescein-labeled S-ODNs was also reported in the neostriatum at 4 h after a bolus intracerebral infusions to rat brain [12]. Nuclear uptake of ODNs and efficiencies of antisense ODNs in vitro are known to be enhanced by encapsulating ODNs with cationic lipids [17]. It remains to be determined whether this is the case in vivo application of anti-
Fig. 2. Photomicrograph,;showing the time course of cellular uptake of ~3P-labeled D- and S-ODNs (15mer) after infusions to mouse brains. (A) D-ODN, 15 rain, (B) D-ODNs at 4 h, (C) S-ODNs at 15 min, (D) S-ODNs at 4 h. Bar is 100 p~m.
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S. Ogawa et al. /Regulatory Peptides 59 (1995) 143-149
Fig. 3. Photomicrographs showing uptake of ~3p-labeled S-ODNs in NF200 (A) or GFAP (B) immunoreactive cells at 4 h after intracerebral infusions. 33P-labeled ODNs were found more frequently in NF200 immuno-positive cells (A), and to a much smaller extent in GFAP immunoreactive cells (B). Bar is 25 /zm. Arrows indicate double-labeled cells.
S. Ogawa et al. /Regulatory Peptides 59 (1995) 143-149
sense ODNs, although it appeared that uptake by the nucleus and nucleolu.,; was more evident with cationic lipid encapsulated ODNs in our preliminary studies (unpublished data). Finally, it was found that ODNs were taken up most avidly by neuronal cells while, to a much smaller extent, both TRITC labeling and silver grain accumulation could he observed in astrocytic cells. These results provide supportive evidence for physiologically significant antisense ODN action on nerve cells.
[8]
[9]
[10]
References [1] Ogawa, S. and Pfaff. D.W., Application of antisense DNA method for the stud) of molecular bases of brain function and behavior, Behav. Genet. (1995) (in press). [2] Wahlestedt, C., Antisense oligonucleotide strategies in neuropharmacology, Trends Pharmacol. Sci., 15 (1994) 42-46. [3] McCarthy, M.M., Kle.opoulos, S.P., Mobbs, C.V. and Pfaff, D.W., Infusion of antisense oligodeoxynucleotides to the oxytocin receptor in the ventromedial hypothalamus reduces estrogen-induced sexual receptivity and oxytocin receptor binding in the female rat, Neuroendocrinology, 59 (1994) 432-440. [4] Standifer, K.M., Chien, C.C., Wahlestedt, C., Brown, G.P. and Pasternak, G.W., Selective loss of delta opioid analgesia and binding by antisense oligodeoxynucleotides to a delta opioid receptor, Neuron, 12 (1994) 805-810. [5] McCarthy, M.M., Masters, D.B., Rimvall, K., SchwartzGiblin, S. and Pfaff, D.W., Intracerebral administration of antisense oligodeoxynucleotides to GAD(65) and GAD(67) mRNAs modulate reproductive behavior in the female rat, Brain Res., 636 (1994) 209-220. [6] Akabayashi, A., Wahlestedt, C., Alexander, J.T. and Leibowitz, S.F., Specific inhibition of endogenous neuropeptide Y synthesis in arcuate nucleus by antisense oligonucleotides suppresses feeding be]aavior and insulin secretion, Mol. Brain Res., 21 (1994) 55-61. [7] McCarthy, M.M., Schleuker, E.H. and Pfaff, D.W., Enduring consequences of neonatal treatment with antisense
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